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In vivo Tn5-based transposon mutagenesis of Streptomycetes

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An Erratum to this article was published on 30 March 2010

Abstract

This paper reports the in vivo expression of the synthetic transposase gene tnp(a) from a hyperactive Tn5 tnp gene mutant in Streptomyces coelicolor. Using the synthetic tnp(a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the Streptomycetes genome. The insertion frequency for the hyperactive Tn5 derivative is 98% of transformed S. coelicolor cells. The random transposition has been confirmed by the recovery of ~1.1% of auxotrophs. The Tn5 insertions are stably inherited in the absence of apramycin selection. The transposon contains an apramycin resistance selection marker and an R6Kγ origin of replication for transposon rescue. We identified the transposon insertion loci by random sequencing of 14 rescue plasmids. The majority of insertions (12 of 14) were mapped to putative open-reading frames on the S. coelicolor chromosome. These included two new regulatory genes affecting S. coelicolor growth and actinorhodin biosynthesis.

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Acknowledgment

We are very grateful to Prof. A. Bechthold for critical discussions during the work as well as for the financial support. The authors also wish to thank Dr. B. Gust for providing plasmid vector pIJ773. Elisabeth Welle is acknowledged for screening of the auxotrophic mutants. Dr. Annette Erb and Stephen Weber are acknowledged for reading and correcting this paper with accuracy.

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  1. Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University of Freiburg, Stefan-Meier st. 19, Freiburg, 79104, Germany

    Lutz Petzke & Andriy Luzhetskyy

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  1. Lutz Petzke
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  2. Andriy Luzhetskyy
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Correspondence to Andriy Luzhetskyy.

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An erratum to this article can be found at https://doi.org/10.1007/s00253-010-2511-9

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Petzke, L., Luzhetskyy, A. In vivo Tn5-based transposon mutagenesis of Streptomycetes. Appl Microbiol Biotechnol 83, 979–986 (2009). https://doi.org/10.1007/s00253-009-2047-z

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  • DOI: https://doi.org/10.1007/s00253-009-2047-z

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